Reversal of propranolol blockade of adrenergic receptors and related toxicity with drugs that increase cyclic AMP.

An overdose of propranolol, a widely used nonselective beta-adrenergic receptor blocking agent, can result in hypotension and bradycardia leading to irreversible shock and death. In addition, the blockade of adrenergic receptors can lead to alterations in neurotransmitter receptors resulting in the interruption of the activity of other second messengers and the ultimate cellular responses. In the present experiment, three agents, aminophylline, amrinone, and forskolin were tested in an attempt to reverse the potential lethal effects of a propranolol overdose in dogs. Twenty-two anesthetized beagle dogs were given a 10-min infusion of propranolol at a dose of 1 mg/kg/min. Six of the dogs, treated only with intravenous saline, served as controls. Within 15-30 min all six control dogs exhibited profound hypotension and severe bradycardia that led to cardiogenic shock and death. Seven dogs were treated with intravenous aminophylline 20 mg/kg 5 min after the end of the propranolol infusion. Within 10-15 min heart rate and systemic arterial blood pressure returned to near control levels, and all seven dogs survived. Intravenous amrinone (2-3 mg/kg) given to five dogs, and forskolin (1-2 mg/kg) given to four dogs, also increased heart rate and systemic arterial blood pressure but the recovery of these parameters was appreciably slower than that seen with aminophylline. All of these animals also survived with no apparent adverse effects. Histopathologic evaluation of the hearts of the dogs treated with aminophylline showed less damage (vacuolization, inflammation, hemorrhage) than the hearts from animals given propranolol alone. Results of this study showed that these three drugs, all of which increase cyclic AMP, are capable of reversing the otherwise lethal effects of a propranolol overdose in dogs.

Reversal of acute theophylline toxicity by calcium channel blockers in dogs and rats.

Theophylline, widely used in the treatment of pulmonary diseases, has a narrow therapeutic index; the recommended plasma levels being 10-20 micrograms/ml in humans. The misuse or abuse of theophylline can cause life-threatening central nervous system and cardiovascular effects. Increased intracellular Ca2+ levels are thought to play an important role in theophylline toxicity and death. The objective of this study was to determine whether Ca2+ channel blockers, e.g. verapamil, nifedipine, or diltiazem, prevent sudden death caused by theophylline treatment in rats and dogs. Groups of Sprague-Dawley rats were treated with theophylline alone (150 mg/kg i.p.) or with theophylline pretreatment followed by administration of verapamil (0.25 to 0.5 mg/kg i.p.), nifedipine (0.25 to 1.0 mg/kg i.p.), or diltiazem (0.5 to 1.0 mg/kg i.p.), 2.5 to 15 min later. The rats were observed for toxic signs and survival over a period of 15 days. All three calcium channel blockers significantly reduced the theophylline-induced sudden death in rats. In a separate study, neither verapamil (0.5 mg/kg i.p.) nor nifedipine (1.0 mg/kg i.p.) prevented the theophylline-induced myocardial necrosis in the rat. In beagle dogs, verapamil (0.5 mg/kg i.v.) prevented theophylline (15 mg/kg/min i.v. for 10 min)-induced hypotension, arrhythmias, and sudden death. Our results support previously reported findings that calcium plays a major role in theophylline-induced toxicity and death.

Synergistic inhibition by benzodiazepine and barbiturate drugs of the clock control of ovulation in hamsters.

A surge of pituitary luteinizing hormone (LH) into the bloodstream occurs in hamsters every 4 days between 1:30 p.m. and 3 p.m. in response to a signal from a biological clock. This surge initiates behavioral estrus approximately 2 h later and ovulation approximately 12 h later. Phenobarbital at a dose > or = 100 mg/kg consistently blocks LH release. Barbiturate and benzodiazepine drugs have separate binding sites in the GABAA receptor/chloride channel complex. Binding of either drug increases GABA-mediated chloride conductance, which suppresses the postsynaptic neuron. Barbiturate binding also increases benzodiazepine binding. This suggested that these drugs might synergize to inhibit LH release. A combination of triazolam and phenobarbital at doses of 10 mg/kg injected s.c. at 1:30 p.m. inhibited ovulation and extended the 4-day vaginal cycle in all treated hamsters. Either drug dose injected alone at 1:30 p.m., or the combination at 3 p.m., was completely ineffective. Bicuculline prevented inhibition by the combination at 1:30 p.m. The clock signal for LH release may act by antagonizing GABA transmission, which may be chronically inhibiting LH release. The combination delimited a 75-min period (1:30-2:45 p.m.) within which the clock signal for LH release occurred in all individuals (ET50 = 2:08 p.m.). This period appears to arise from individuals with different but constant clock settings rather than from a 75-min variation in the clock setting of the individual.

Enhancement of acute myocardial lesions by asthma drugs in rats.

Asthma morbidity and mortality have risen significantly in the last 10 years. The reasons for the increase are multifactorial. One proposed explanation is possible myocardial toxicity arising from the use of beta-agonists alone or in combination with methylxanthines. Previous studies have shown that beta-agonists given alone and beta-agonist/methylxanthine combinations given at higher than recommended clinical doses induced dose-related cardiotoxicity and sudden death in rats. The objective of the present study was to determine whether or not beta-agonists given alone and in combination with methylxanthines at recommended clinical doses also induce cardiotoxicity and sudden death in rats. The beta-agonists, isoproterenol hydrochloride (15 micrograms/kg), fenoterol hydrobromide (40 micrograms/kg), and terbutaline hemisulfate (0.4 mg/kg) were given in single sc doses separately and concurrently with the methylxanthines aminophylline hydrate (20 mg/kg) and caffeine (40 mg/kg), which were given up to a susceptible animal model, the heavy Sprague-Dawley rat. beta-agonist-induced myocardial toxicity (necrosis) was observed. The toxicity was enhanced by aminophylline resulting in the sudden death (most likely due to ventricular fibrillation) of some animals. A decrease in serum iron levels was observed in rats of all beta-agonist and/or methylxanthine-treated groups.

Brain neurotransmitter receptor alterations in offspring of rats exposed to phenobarbital, phenytoin or their combination during pregnancy.

Neurotransmitter receptor binding was studied in the progeny of rats exposed to phenobarbital (80 mg/kg), phenytoin (80 mg/kg) or their combination daily from 5 to 20 days of gestation. At 22 days postnatally, female pups in all treated groups showed decreased binding of [3H]spiroperidol to striatal membranes. A decreased binding of [3H]diazepam to frontal cortical membranes was observed in the female pups of rats exposed to phenytoin or the combination. The Scatchard analysis of data from the affected groups revealed a decrease in the maximum number of dopamine and benzodiazepine receptor sites and no change in their affinities. The binding of [3H]spiroperidol or [3H]diazepam was not altered significantly in any of the drug-treated male pups. Also, no significant change in the binding of [3H]serotonin or [3H]quinuclidinyl benzilate to frontal cortical membranes was observed in any of the treated groups. At 60 days postnatally, no significant change in the binding of any of the above ligands was evident. The above prenatal drug exposures appear to cause transitory subsensitivity of dopamine and benzodiazepine receptors in rats.

Delayed development of reproductive functions and alteration of dopamine receptor binding in hypothalamus of rats exposed prenatally to phenytoin and phenobarbital.

Previous work in experimental animals treated prenatally with phenobarbital revealed reproductive changes in the offspring. Little has been reported on the effect of phenytoin or a combination of both drugs. 75 Pregnant Sprague-Dawley rats were administered either phenytoin (Na salt) at 20, 40 or 80 mg/kg orally (p.o.), phenobarbital (Na salt) at 20, 40 or 80 mg/kg subcutaneously (s.c.) or a combination of both drugs at the same doses daily from 5 to 20 days of pregnancy (4-12 rats/dose level). 24 Control rats received 1% methylcellulose p.o. and distilled water s.c. Regularity of estrous cycles was decreased in all treated groups, with the phenytoin group being most affected. The binding of [3H]spiroperidol, known to label dopamine receptors, was decreased in the hypothalamus of all drug-treated groups, indicating that the alteration in neuroendocrine function may be responsible for the above effects. Data suggest that prenatal exposure of rats to anticonvulsant drugs could lead to reproductive disorders.

Macromolecular levels, DNA synthesis and ornithine decarboxylase activity in leg muscles from 6-mercaptopurine-treated rats.

Sprague-Dawley male and female rats were treated with 6-mercaptopurine (6-MP) (2 mg/kg sc) daily from 2 to 22 days of age and killed at 7, 15, 27 and 64 days of age. At 7 and 27 days of age rats were injected with 3H thymidine for measurement of DNA synthesis. Fore- and hindlimb muscles were removed and analyzed for ornithine decarboxylase (ODC) activity (all ages), DNA radioactivity (7 and 27 days), DNA level (27 and 64 days) and RNA level (64 days). As expected, ODC activity and DNA synthesis were higher in muscles of 7-day-old rats than in muscles of the older rats studied. A consistently lower ODC activity was seen in 6-MP-treated vs. control rats for 5-25 days after start of treatment, but the effect was essentially the same for the hindlimb and forelimb muscles. During the 7-27-day time course ODC activity was higher in hindlimb than forelimb muscles. By 27 days of age DNA synthesis was also higher in the hindlimb muscles. DNA synthesis was decreased after 5 days of treatment relative to that of control rats, to an approximately equal extent in forelimb and hindlimb muscles. Five days after the last treatment a trend was seen for slower recovery from inhibition of DNA synthesis in hindlimb muscles, particularly in male rats. DNA levels were reduced in treated rats relative to those in control rats 5 days after the last treatment to approximately the same degree in forelimb and hindlimb muscles. Forty-two days after the last treatment a trend toward increased activity of ODC and increased DNA and RNA levels was seen in muscles of treated rats, probably reflective of recovery processes. These early biochemical effects of 6-MP, which were seen to about the same extent in the forelimb and hindlimb muscles cannot explain by themselves the delayed hindlimb fat atrophy resulting from 6-MP treatment of neonatal rats.

Toxicological studies with 6-mercaptopurine in neonates.

In previous studies, we found that Sprague-Dawley rats injected with 6-MP monohydrate at 2 mg base/kg sc daily from 2 to 22 days of age had atrophy of thigh and sublumbar muscles when killed at 16 months of age. The first sign of this muscle atrophy was detected grossly (flattened croup with or without paresis) at 12 months of age. In one experiment of the present work, using the same treatment in rats as above, we found that the earliest onset of muscle atrophy observed by light microscopy occurred at 2 months of age. By 4 months the atrophy could be detected grossly. The atrophy did not uniformly involve all muscles of the hindquarters; the thigh (especially the semitendinosus), leg (soleus but not the extensor carpi group), and lumbar vertebral (including the psoas) muscles were involved. Foreleg (biceps), intercostal, and tongue muscles as well as the sciatic nerve and internal organs appeared unaffected. In another experiment, weanling Sprague-Dawley rats given large daily doses of 6-MP from 25 to 45 days of age had normal muscles when killed at 8 months. In a third experiment, Wistar rats injected with 6-MP (2 mg base/kg sc) daily from 2 to 22 days of age and killed at 6 months had muscle atrophy similar to that seen in Sprague-Dawley rats. In the last experiment, mice and hamsters given large daily doses of 6-MP from 2 to 22 days of age had normal muscles when killed at 10 months. It appears from these results that the 6-MP-induced muscle atrophy occurs only after treatment during the neonatal period and that the atrophy may be species-specific.

Muscle atrophy and histopathology of the soleus in 6-mercaptopurine-treated rats.

In this study, male and female Sprague-Dawley rats were treated neonatally with 6-MP-treatment (2 mg/kg s/c, between 2 and 22 days after birth) and evaluated at six months of age. Compared to the normal controls, the 6-MP-treated male and female rats showed similar sciatic nerve conduction to the soleus. However, there was a significant muscle atrophy (57-60%, P less than 0.01) and a decrease in areas of the type I (42-54%, P less than 0.05) and type II (41-71%, P less than 0.01) muscle fibers. The number of type II fibers declined significantly (7.4-14.8%, P less than 0.05). It is proposed that the soleus muscle atrophy and histopathology in 6-MP-treated rats is unrelated to nerve conduction defects and may be related to growth inhibition caused by an interference of the drug during normal differentiation of muscle fibers.

Ultrastructural changes in skeletal muscles of rats treated neonatally with 6-mercaptopurine.

The purpose of this study was to characterize the ultrastructural changes in the muscles of 2- and 6-month-old rats that were injected with 6-mercaptopurine monohydrate (6-MP) at 2 mg base/kg sc daily from 2 to 22 days of age. The earliest, usually subsarcolemmal alterations, were disorganization of fibrils at the Z-bands with excessive Z-band-like material and loss of myofibrils. In the most extensive lesions, the entire fiber was involved, with vacuoles within the disorganized fibrils. The peripheral nerves and vessels were unremarkable. Thus 6-MP-induced changes appear to be primarily myogenic in origin.

6-Mercaptopurine treatment affects the membrane potentials of rat skeletal muscle fibers.

6-Mercaptopurine (6-MP) was injected daily (2 mg/kg sc) into 24 Sprague-Dawley rats during the first three weeks of life. There were 23 saline-injected control animals. Atrophy of the muscles of the hindquarters in the 6-MP-treated rats began at about 4 months of age. The membrane potentials (Em) of the isolated extensor digitorum longus (EDL) and the caudofemoralis (CF) muscle (in situ) were measured with intracellular microelectrodes at 6-18 months of age. It was found that there was a wide spectrum of fibers with respect to electrical abnormalities in the 6-MP-treated muscles, some fibers having perfectly normal parameters. However, the mean resting Em of fibers in the EDL muscle of 6-MP-treated rats (-61.1 +/- 0.7 mV) was lower than that of the control rats (-69.7 +/- 0.6 mV). The same was true for the fibers of the CF muscle (-64.9 +/- 1.5 mV for 6-MP-treated fibers vs -71.6 +/- 1.3 mV for controls). Experiments done in the presence and absence of ouabain indicated that the contribution of the electrogenic pump potential to Em was similar in 6-MP-treated and control rats, and therefore could not account for the depolarization observed in 6-MP-treated rats. The data also demonstrated that this depolarization was not due to a decreased intracellular K+ concentration. The Na+/K+ permeability ratio (PNa/PK) was higher in the 6-MP-treated rats, and could account for the decreased resting Em. The APs of 6-MP-treated rats (elicited from the natural Em of the fiber) had more fibers with a lower maximum rate of rise (+Vmax) (330 +/- 20 vs 391 +/- 17 V/sec) and lower amplitude (65.1 +/- 2.9 vs 73.3 +/- 1.8 mV) than in the control muscles. When hyperpolarized to -90 mV before eliciting the AP, fibers from 6-MP-treated rats still displayed depressed AP rates of rise (+Vmax of 382 +/- 19 vs 511 +/- 21 V/sec in controls), depressed AP amplitudes (97 +/- 2.1 vs 105 +/- 1.6 mV in controls) and slightly prolonged duration at 50% amplitude (APD50) (0.66 +/- 0.03 vs 0.60 +/- 0.02 sec in controls). These electrophysiological alterations produced by this chemically-induced myopathy are similar to those observed in murine muscular dystrophy.

Effects of 6-mercaptopurine treatment on the membrane potentials of rat skeletal muscle fibers.

6-Mercaptopurine (6-MP), injected daily (2 mg/kg s.c.) into Sprague-Dawley rats during the first 3 weeks of life, causes atrophy in muscles of the hindquarters beginning at 4 months of age. The extensor digitorium longus (EDL) muscles from 24 rats injected with 6-MP and 23 saline-injected controls, 6-18 months of age, were studied. Electron microscopy showed a number of abnormalities in the EDL muscle of 6-MP-treated rats, such as myocytes with atypical ultrastructure (including disorganized myofibrils) adjacent to structurally normal cells. Membrane potentials (Em) were measured in the isolated EDL and in the caudofemoralis (CF) muscle in situ. The mean Em of fibers in the EDL of 6-MP-treated rats (-61.1 +/- 0.7 (SE) mV) was lower than that of the control rats (-69.7 +/- 0.6 mV). The same was true for the fibers of the CF muscle (-64.9 +/- 1.5 mV for 6-MP-treated fibers vs. -71.6 +/- 1.3 mV for controls). The contribution of the electrogenic pump potential to Em (+/- ouabain) was similar in 6-MP-treated and control rats, and therefore could not account for the depolarization observed in 6-MP-treated rats. This depolarization was not due to a decreased intracellular K+ concentration. The Na+:K+ permeability ratio (PNa/PK) was higher in the 6-MP-treated rats and could account for the decrease in Em.(ABSTRACT TRUNCATED AT 250 WORDS)

Muscle atrophy and histopathology of the soleus in 6-mercaptopurine-treated rats.

This study was conducted to examine the histochemical changes occurring due to neonatal 6-mercaptopurine (6-MP) treatment (2 mg/kg, sc, between 2 and 22 days after birth) in the slow-twitch muscle (soleus) of adult male and female rats. At 6 months of age, the control and the 6-MP-treated rats were evaluated for the sciatic nerve conduction to the soleus and for the soleus atrophy and histopathology of the type I (slow-twitch) and type II (fast-twitch) muscle fibers. Compared to the normal controls, the 6-MP-treated male and female rats showed similar sciatic nerve conduction to the soleus. However, there was a significant muscle atrophy (57-60%, P less than 0.01) and a decrease in fiber areas of the type I (42-54%, P less than 0.05) and type II (41-71%, P less than 0.01) fibers. The number of type II fibers declined significantly (7.4-14.8%, P less than 0.05). It is proposed that the soleus muscle atrophy and histopathology in 6-MP-treated rats is unrelated to nerve conduction defects and may be related to growth inhibition caused by an interference of the drug during normal differentiation of muscle fibers.

Further studies on 6-mercaptopurine-induced muscle atrophy in rats, mice, and hamsters treated as neonates.

In previous studies, we found that Sprague-Dawley rats injected with 6-mercaptopurine monohydrate (6-MP) at 2 mg base/kg sc daily from 2 to 22 days of age had atrophy of thigh and sublumbar muscles when killed at 16 months of age. The first sign of this muscle atrophy was detected grossly (flattened croup with or without paresis) at 12 months of age. In one experiment of the present work, using the same treatment in rats as above, we found that the earliest onset of muscle atrophy observed by light microscopy occurred at 2 months of age. By 4 months the atrophy could be detected grossly. The atrophy did not uniformly involve all muscles of the hindquarters; the thigh (especially the semitendinosus), leg (soleus but not the extensor carpi group), and lumbar vertebral (including the psoas) muscles were involved. Foreleg (biceps), intercostal, and tongue muscles as well as the sciatic nerve and internal organs appeared unaffected. In another experiment, weanling Sprague-Dawley rats given large daily doses of 6-MP from 25 to 45 days of age had normal muscles when killed at 8 months. In a third experiment, Wistar rats injected with 6-MP (2 mg base/kg sc) daily from 2 to 22 days of age and killed at 6 months had muscle atrophy similar to that seen in Sprague-Dawley rats. In the last experiment, mice and hamsters given large daily doses of 6-MP from 2 to 22 days of age had normal muscles when killed at 10 months. It appears from these results that the 6-MP-induced muscle atrophy occurs only after treatment during the neonatal period and that the atrophy may be species specific.

Alteration of high-affinity binding sites of neurotransmitter receptors in rats after neonatal exposure to streptomycin.

Central neurotransmitter functions were analyzed in dyskinetic rats that were treated subacutely with streptomycin as neonates. Data indicated that the treatment caused an increase in the binding of [3H]spiroperidol in striatum and of [3H]serotonin in frontal cortex, but no significant changes were observed in the binding of [3H]quinuclidinyl benzilate and [3H]muscimol to cerebellum and of [3H]diazepam to frontal cortex. The increase in the binding of [3H]spiroperidol and [3H]serotonin was evident in both sexes; however, the increase of [3H]spiroperidol binding was statistically significant only in females. Kinetic studies as revealed by Scatchard plots show that the increase in the binding of the two ligands is due to an increase in the number of the receptors. These results support the concept of a central mechanism of action for the streptomycin-induced dyskinesias.

Muscular degeneration in rats after postnatal treatment with 6-mercaptopurine.

6-Mercaptopurine monohydrate was injected sc at 2 mg base/kg/day from 2 to 22 days of age to four litters of rat pups (four females, four males per litter). Control neonates were injected sc with basic saline (pH 8). Daily observations for signs of toxicity were made during the treatment period and once weekly thereafter until the rats were 6 months of age. The pups were weighed at 2, 12, 23, 34, 100, and 480 days of age. Fertility was tested at 3 to 6 months of age. From 6 months of age on, the rats were examined for tumors at 3-month intervals until the experiment was terminated at 16 months of age. A reduction in body weight of treated rats began between 34 and 100 days of age and became more pronounced by 16 months of age. Fertility was similar in treated and control groups and there were no detectable tumors in either group. The major finding in treated rats was a delayed onset of hind leg paresis that was first detected at 12 months of age. Light microscopic examination of tissues taken from the hind quarters of these rats at 16 months of age revealed a severe atrophic degeneration with fatty infiltration of sublumbar and thigh muscles.